Toral Zaveri

Contributions of surface topography and cytotoxicity to the macrophage response to zinc oxide nanorods.

Macrophages associated with implanted biomaterials are primary mediators of chronic inflammation and foreign body reaction to the implant. Hence, various approaches have been investigated to modulate macrophage interactions with biomaterial surfaces to mitigate inflammatory responses. Nanostructured materials possess unique surface properties, and nanotopography has been reported to modulate cell adhesion and viability in a cell type-dependent manner. Zinc oxide (ZnO) has been investigated in a number of biomedical applications and surfaces presenting well-controlled nanorod structures of ZnO have recently been developed. In order to investigate the influence of nanotopography on macrophage adhesive response, we evaluated macrophage adhesion and viability on ZnO nanorods, compared to a relatively flat sputtered ZnO controls and using glass substrates for reference. We found that although macrophages are capable of initially adhering to and spreading on ZnO nanorod substrates, the number of adherent macrophages on ZnO nanorods was reduced compared to ZnO flat substrate and glass. Additionally adherent macrophage number on ZnO flat substrate was reduced as compared to glass. While these data suggest nanotopography may modulate macrophage adhesion, reduced cell viability on both sputtered and nanorod ZnO substrate indicates appreciable toxicity associated with ZnO. Cell death was apparently not apoptotic, given the lack of activated caspase-3 immunostaining. A decrease in viable macrophage numbers when ZnO substrates were present in the same media verified the role of ZnO substrate dissolution, and dissolved levels of Zn in culture media were quantified. In order to determine long-term physiological responses, ZnO nanorod-coated and sputtered ZnO-coated polyethylene terephthalate (PET) discs were implanted subcutaneously in mice for 14 d. Upon implantation, both ZnO-coated discs resulted in a discontinuous cellular fibrous capsule indicative of unresolved inflammation, in contrast to uncoated PET discs, which resulted in typical foreign body capsule formation. In conclusion, although ZnO substrates presenting nanorod topography have previously been shown to modulate cellular adhesion in a topography-dependent fashion for specific cell types, this work demonstrates that for primary murine macrophages, cell adhesion and viability correlate to both nanotopography and toxicity of dissolved Zn, parameters which are likely interdependent.

Microparticle surface modifications targeting dendritic cells for non-activating applications

Microparticulate systems for delivery of therapeutics to DCs for immunotherapy have gained attention recently. However, reports addressing the optimization of DC-targeting microparticle delivery systems are limited, particularly for cases where the goal is to deliver payload to DCs in a non-activating fashion. Here, we investigate targeting DCs using poly (d lactide-co-glycolide) microparticles (MPs) in a non-stimulatory manner and assess efficacy in vitro and in vivo. We modified MPs by surface immobilizing DC receptor targeting molecules - antibodies (anti-CD11c, anti-DEC-205) or peptides (P-D2, RGD), where anti-CD11c antibody, P-D2 and RGD peptides target integrins and anti-DEC-205 antibody targets the c-type lectin receptor DEC-205. Our results demonstrate the modified MPs are neither toxic nor activating, and DC uptake of MPs in vitro is improved by the anti-DEC-205 antibody, the anti-CD11c antibody and the P-D2 peptide modifications. The P-D2 peptide MP modification significantly improved DC antigen presentation in vitro both at immediate and delayed time points. Notably, MP functionalization with P-D2 peptide and anti-CD11c antibody increased the rate and extent of MP translocation in vivo by DCs and MΦs, with the P-D2 peptide modified MPs demonstrating the highest translocation. This work informs the design of non-activating polymeric microparticulate applications such as vaccines for autoimmune diseases.

Integrin-directed modulation of macrophage responses to biomaterials

Macrophages are the primary mediator of chronic inflammatory responses to implanted biomaterials, in cases when the material is either in particulate or bulk form. Chronic inflammation limits the performance and functional life of numerous implanted medical devices, and modulating macrophage interactions with biomaterials to mitigate this response would be beneficial. The integrin family of cell surface receptors mediates cell adhesion through binding to adhesive proteins nonspecifically adsorbed onto biomaterial surfaces. In this work, the roles of integrin Mac-1 (αMβ2) and RGD-binding integrins were investigated using model systems for both particulate and bulk biomaterials. Specifically, the macrophage functions of phagocytosis and inflammatory cytokine secretion in response to a model particulate material, polystyrene microparticles were investigated. Opsonizing proteins modulated microparticle uptake, and integrin Mac-1 and RGD-binding integrins were found to control microparticle uptake in an opsonin-dependent manner. The presence of adsorbed endotoxin did not affect microparticle uptake levels, but was required for the production of inflammatory cytokines in response to microparticles. Furthermore, it was demonstrated that integrin Mac-1 and RGD-binding integrins influence the in vivo foreign body response to a bulk biomaterial, subcutaneously implanted polyethylene terephthalate. A thinner foreign body capsule was formed when integrin Mac-1 was absent (~30% thinner) or when RGD-binding integrins were blocked by controlled release of a blocking peptide (~45% thinner). These findings indicate integrin Mac-1 and RGD-binding integrins are involved and may serve as therapeutic targets to mitigate macrophage inflammatory responses to both particulate and bulk biomaterials.

User preferences in a carrageenan-based vaginal drug delivery system.

Topical microbicides are a promising solution to address the global threat of HIV and other sexually transmitted infections. To be successful, a microbicide not only needs to be biologically functional but also highly acceptable to users. User acceptability of microbicides can be incorporated early in the product formulation and design process. Previous qualitative research revealed women had strong preferences regarding product shape, while preferences related to size and firmness were less clear. Here, we explored the effect of size and firmness on the acceptability of semisolid ovoid microbicide prototypes intended for vaginal use. Sexually active women (n = 74) were randomized to one of two conditions: with and without applicator. Nine different prototypes were evaluated; they were formulated to low, medium and high firmness using mixtures of kappa and iota carrageenan and potassium chloride. Three sizes were produced at each firmness level. Women manipulated all nine prototypes, rating them for perceived effectiveness, imagined ease-of-insertion and willingness-to-try on visual analog scales. The influence of size and firmness on these three outcome measures were assessed using ANOVA and response surface models. Results indicated size and firmness both influenced the outcome measures, but firmess was more influential than size. Also, the specific effects of size and firmness depended strongly on presence or absence of an applicator. Generally, women in the without applicator condition wanted a larger, firmer product. Collectively, these data suggest efforts to rationally design of microbicides for enhanced user acceptability must consider factors like size and firmness. Also, the decision to include or forego an applicator should be addressed early in the design process, as it strongly influences other design decisions.

Release of tenofovir from carrageenan-based vaginal suppositories.

Microbicides are an active area of research for HIV prevention, being developed as a woman-initiated method of prevention during unprotected coitus. Along with safety and efficacy, assessing and improving compliance is a major area of research in microbicide development. We have produced microbicide prototypes in the form of semisoft vaginal suppositories prepared from carrageenan and conducted both qualitative and quantitative studies using these prototypes to determine the physical properties that drive acceptability and possibly adherence. In order to ensure that the suppositories function as effective drug delivery vehicles, we have conducted in vitro dissolution studies in water, vaginal simulant fluid (VSF) and semen simulant fluid (SSF) with suppositories loaded with the antiretroviral drug, tenofovir (TFV). TFV was released via diffusion and matrix erosion in water or by diffusion out of the matrix in VSF and SSF. Diffusion studies were conducted in two different volumes of VSF and SSF. The volume of VSF/SSF into which TFV diffused and the size of the suppositories determined the rate of diffusion from the suppositories. About 45%–50% of the encapsulated TFV diffused out of the suppositories within the first two hours, irrespective of suppository size, diffusion medium (VSF/SSF) and the volume of medium. Prior work indicates that a short waiting period between insertion and coitus is highly desired by women; present data suggest our microbicide prototypes have rapid initial release followed by a slow release curve over the first 24 h.

Relationships between Perceptual Attributes and Rheology in Over-the-Counter Vaginal Products: A Potential Tool for Microbicide Development

Vaginal microbicides are believed to have substantial potential to empower women to protect themselves from HIV, although clinical trials to date have had mixed results at best. Issues with patient adherence in these trials suggest additional emphasis should be placed on optimizing acceptability. Acceptability is driven, in part, by the sensory properties of the microbicide, so better understanding of the relationships between sensory properties and the physical and rheological properties of microbicides should facilitate the simultaneous optimization of sensory properties in parallel with the biophysical properties required for drug deployment. Recently, we have applied standard methods to assess the potential acceptability of microbicide prototypes ex vivo and to quantify the sensory properties of microbicide surrogates. Here, we link quantitative perceptual data to the rheological properties of 6 over-the counter (OTC) vaginal products used as ex vivo microbicide surrogates. Shear-thinning behavior (n) and tan δ (10 rad/s) showed no relationship with any perceptual attributes while shear storage modulus, G’ (10 rad/s) was correlated with some attributes, but did not appear to be a strong predictor of sensory properties. Conversely, the storage loss modulus, G” (10 rad/s) and the consistency coefficient, K, were correlated with several sensory attributes: stickiness, rubberiness, and uniform thickness for G’’ and stickiness, rubberiness, and peaking for K. Although these relationships merit confirmation in later studies, this pilot study suggests rheological principles can be used to understand the sensory properties evoked by microbicide surrogates assessed ex vivo. Additional work is needed to determine if these findings would apply for microbicides in vivo.

Firmness Perception Influences Women's Preferences for Vaginal Suppositories.

Microbicides are being actively researched and developed as woman-initiated means to prevent HIV transmission during unprotected coitus. Along with safety and efficacy, assessing and improving compliance is a major area of research in microbicide development. We have developed carrageenan-based semisoft vaginal suppositories and have previously evaluated how physical properties such as firmness, size and shape influence women’s willingness to try them. Firmness has previously been quantified in terms of small-strain storage modulus, G’, however large-strain properties of the gels may also play a role in the firmness perception. In the current study we prepared two sets of suppositories with the same G’ but different elongation properties at four different G’ values (250, 2500, 12,500, 25,000 Pa): For convenience we refer to these as “brittle” and “elastic”, although these terms were never provided to study participants. In the first of two tests conducted to assess preference, women compared pairs of brittle and elastic suppositories and indicated their preference. We observed an interaction, as women preferred brittle suppositories at lower G’ (250, 2500 Pa) and elastic ones at a higher G’ (25,000 Pa). In the second test, women evaluated samples across different G’, rated the ease-of-insertion and willingness-to-try and ranked the samples in order of preference. Brittle suppositories at G’ of 12,500 Pa were most preferred. In vitro studies were also conducted to measure the softening of the suppositories in contact with vaginal simulant fluid (VSF). Release of antiretroviral drug tenofovir in VSF was quantified for the brittle and elastic suppositories at G’ of 12,500 Pa to determine the effect of suppository type on release. The initial rate of release was 20% slower with elastic suppositories as compared to brittle suppositories. Understanding how different physical properties simultaneously affect women’s preferences and pharmacological efficacy in terms of drug release is required for the optimization of highly acceptable and efficacious microbicides.

Drivers of Vaginal Drug Delivery System Acceptability from Internet-Based Conjoint Analysis

Vaginal microbicides potentially empower women to protect themselves from HIV and other sexually transmitted infections (STIs), especially when culture, religion, or social status may prevent them from negotiating condom use. The open literature contains minimal information on factors that drive user acceptability of women’s health products or vaginal drug delivery systems. By understanding what women find to be most important with regard to sensory properties and product functionality, developers can iteratively formulate a more desirable product. Conjoint analysis is a technique widely used in market research to determine what combination of elements influence a consumer’s willingness to try or use a product. We applied conjoint analysis here to better understand what sexually-active woman want in a microbicide, toward our goal of formulating a product that is highly acceptable to women. Both sensory and non-sensory attributes were tested, including shape, color, wait time, partner awareness, messiness/leakage, duration of protection, and functionality. Heterosexually active women between 18 and 35 years of age in the United States (n = 302) completed an anonymous online conjoint survey using IdeaMap software. Attributes (product elements) were systematically presented in various combinations; women rated these combinations of a 9-point willingness-to-try scale. By coupling systematic combinations and regression modeling, we can estimate the unique appeal of each element. In this population, a multifunctional product (i.e., broad spectrum STI protection, coupled with conception) is far more desirable than a microbicide targeted solely for HIV protection; we also found partner awareness and leakage are potentially strong barriers to use.

Macrophage Response to Zinc Oxide Nanorod Surfaces -Topography and Toxicity

Macrophages recruited to the site of biomaterial implantation are the primary mediators of the chronic foreign body response to implanted materials. Hence various approaches have been investigated to modulate macrophage interactions with biomaterial surfaces to mitigate the inflammatory response. The extent of the inflammatory response mounted by the body has been shown to be modulated by the implant material and its surface properties. Because of their unique surface properties, there is great interest in exploring nanostructured materials for potential biomedical applications. Nanotopography is known to modulate cell adhesion and viability depending on cell type. As macrophage adhesion to biomaterial surfaces is one of the first steps of the inflammatory response to a foreign body, a surface which inhibits macrophage adhesion and function may serve to modulate the foreign body response. ZnO is used in a number of biomedical applications such as glucose detection, wound healing and dental filling materials. We have evaluated macrophage adhesion and viability on ZnO nanorods as compared to a control sputtered ZnO flat substrates and reference glass surfaces. Macrophage adhesion and viability was reduced by 50 % on nanorods as compared to ZnO flat substrate and glass suggesting that this nanotopography is potentially useful for biomaterial surfaces. Macrophage viability decreased when ZnO nanorods [62%] and flat surfaces [45%] were present in the same media but not in cell contact, suggesting moderate levels of toxicity associated with ZnO due to dissolution in media. The decreased viability when macrophages were in contact compared to non-contact suggests that modulation of cell adhesion and viability on nanorods is dominantly due to topography rather than material toxicity. We therefore plan to further investigate these nanotopographies constructed from other materials which are not toxic to macrophages in order to explore macrophage-modulating surfaces for biomaterial applications.

Qualitative exploration of intrinsic and extrinsic factors that influence acceptability of semisoft vaginal suppositories

BackgroundVaginal microbicides are a promising means to prevent the transmission of HIV and other sexually transmitted infections, by empowering women to initiate use prophylactically when they perceive themselves to be at risk. However, in clinical trials, microbicides have shown mixed results, with the consistent finding that effectiveness varies substantially as a function of user adherence.MethodsBased on the assumption that adherence is driven, at least in part, by product properties that influence acceptability, we used softgel technology to develop vaginal drug delivery systems in the intermediate texture space between solids and liquids to overcome potential shortcomings of current dosage forms. Here, we used focus groups and surveys to determine women’s initial reactions (i.e., acceptance and willingness-to-try) for semisoft vaginal suppositories intended for HIV and STI prevention, with a specific focus on how perception of and preferences for vaginal suppositories may be influenced by product characteristics such as size, shape, and firmness.ResultsVia focus groups, we identified intrinsic and extrinsic factors relevant to acceptability of semisoft suppository prototypes. Willingness-to-try depended on factors like intended functionality, anticipated leakage, type of sex, recommended frequency of use, type of sexual partner, and perceived risk. When handled ex vivo, shape, size, and firmness of suppositories communicated information about ease of imagined insertion and handling, perceived effectiveness, anticipated awareness and comfort of the product in the body. These impressions were partly based on prior experience with vaginal products.ConclusionsSensory attributes appear to play a substantial role in women’s preferences and willingness to try the semisoft suppositories. Using these methods during preclinical development should help efficiently optimize a final product that is both biologically efficacious and preferred by women, toward a goal of enhancing adherence and effectiveness.